Summary

Although patients with Parkinson’s disease show impairments in cognitive performance even at the early stage of the disease, the synaptic mechanisms underlying cognitive impairment in this pathology are unknown. Hippocampal long-term potentiation represents the major experimental model for the synaptic changes underlying learning and memory and is controlled by endogenous dopamine. We found that hippocampal long-term potentiation is altered in both a neurotoxic and transgenic model of Parkinson’s disease and this plastic alteration is associated with an impaired dopaminergic transmission and a decrease of NR2A/NR2B subunit ratio in synaptic N-methyl-d-aspartic acid receptors. Deficits in hippocampal-dependent learning were also found in hemiparkinsonian and mutant animals. Interestingly, the dopamine precursor l-DOPA was able to restore hippocampal synaptic potentiation via D1/D5 receptors and to ameliorate the cognitive deficit in parkinsonian animals suggesting that dopamine-dependent impairment of hippocampal long-term potentiation may contribute to cognitive deficits in patients with Parkinson’s disease.

α-synuclein

CA1 area

dementia

dopamine

glutamate

synaptic plasticity

Introduction

Parkinson’s disease causes impairments in cognitive performance resembling those seen in frontal lobe patients (Robbins and Arnsten, 2009), and progression of these deficits can lead to dementia (Cools et al., 2001; Goetz et al., 2008). While the motor abnormalities in Parkinson’s disease result from nigrostriatal dopamine depletion, memory dysfunctions are induced by degeneration of the dopaminergic mesocorticolimbic projections originating from the ventral tegmental area (Calabresi et al., 2006).

The hippocampus is essential for encoding spatial and episodic memories (Ryan et al., 2010), and for novelty detection (Jenkins et al., 2004). Novelty detection involves the comparison of an existing memory with new sensory information. Indeed, CA1 pyramidal cells also receive direct sensory inputs from the cortex (Lisman and Otmakhova, 2001). The dopaminergic system is a strong candidate for modulating novelty acquisition and synaptic plasticity in the hippocampal CA1 area. The ventral tegmental area, the major brain source of dopaminergic inputs to the limbic system and hippocampus, is part of a functional loop detecting novelty (Lisman and Grace, 2005).

Intracellular accumulation of α-synuclein is the pathological feature of Parkinson’s disease and monogenic forms of this disease have been associated with mutations in the gene encoding for this protein (Dawson et al., 2010). Although abnormalities in hippocampal plasticity have been observed in α-synuclein-related models (Steidl et al., 2003; Gureviciene et al., 2009), the mechanisms underlying the alteration of hippocampal LTP and hippocampal-related memory have never been analysed and compared in a toxic and genetic model of Parkinson’s disease. To address this issue, we investigated hippocampal dopamine transmission and LTP in 6-hydroxydopamine (6-OHDA) hemilesioned rats and α-synuclein transgenic mice, expressing a truncated form of human α-synuclein (1–120; Tofaris et al., 2006).

Materials and methods

Rats with 6-hydroxydopamine-induced lesion

Three-month-old 6-OHDA-lesioned rats (n = 81) were obtained as previously reported (Picconi et al., 2003, 2008). Briefly, a group of deeply anaesthetized (chloral hydrate, 400 mg/ml/kg) rats were injected unilaterally with 6-OHDA (12 µg/4 µl of saline containing 0.1% ascorbic acid) into the medial forebrain bundle at a rate of 0.38 µl/min (anterioposterior = −4.4, lateral = +1.2, ventrodorsal =−7.8). A group of rats were injected only with vehicle at the same coordinates (sham-operated rats; n = 43). Fifteen days later, rats were tested with 0.05 mg/kg subcutaneous apomorphine, and contralateral turns to the lesion were counted for 40 min. The rats that showed more than 200 contralateral turns were enrolled in the study. Sham-operated animals did not show any turning behaviour. The severity of the lesion was also quantified afterward by nigral tyrosine hydroxylase immunohistochemistry. Experiments were performed 4–6 weeks after lesion.

Mice transgenic for truncated human α-synuclein (1–120)

Male mice (3–4 months old) transgenic for truncated human α-synuclein (1–120; n = 23), produced on a C57BL/6S background (Harlan; Tofaris et al., 2006), and control aged-matched C57BL/6S male wild-type mice which is a strain of C57Bl/6 that lacks endogenous α-synuclein (Harlan; n = 18), were used in electrophysiological experiments, dopamine tissue quantification, molecular analysis and for behavioural testing. In these transgenic mice (α-syn120), the expression of truncated human α-synuclein (1–120), driven by the tyrosine hydroxylase promoter on a mouse α-synuclein null background, leads to the formation of pathological inclusions in the substantia nigra and olfactory bulb and to the reduction in striatal dopamine levels. At the behavioural level, the transgenic mice show a progressive reduction in spontaneous locomotion.

Immunohistological procedure

In 6-OHDA rats, the severity of the lesion was confirmed afterward by tyrosine hydroxylase immunohistochemistry. For each rat, tyrosine hydroxylase-positive cells were counted at three different rostrocaudal levels of the substantia nigra compacta at the level of the exiting of the third nerve, 200 µm rostral and 200 µm caudal to this level. Cell number was expressed as the mean number/section and the loss of tyrosine hydroxylase-positive cells was analysed by two-way ANOVA, followed by Tukey’s post hoc test.

Behavioural procedure

The protocol used for the hole-board test is a modified version of that described by Kemp and Manahan-Vaughan (2008). Horizontal and vertical movements were recorded in an automated apparatus (Imetronic). The hole-board test was performed in two different experimental sections: (i) 6-OHDA-lesioned and sham-operated rats, α-synuclein and wild-type mice and (ii) hemiparkinsonian rats, α-synuclein mice and their relative controls, subchronically treated with l-DOPA. ANOVA was used to analyse statistical differences between groups, and Tukey’s Honestly Significant Difference test for post hoc comparisons.

Synaptosome preparation and [3H]-dopamine analysis

Synaptosomes were prepared as previously described (Marti et al., 2003). Briefly, hippocampus was homogenized in ice-cold 0.32M sucrose buffer (pH 7.4), then centrifuged for 10 min at 2500gmax (4°C). The supernatant was centrifuged for 20 min at 9500gmax (4°C), and the synaptosomal pellet resuspended in oxygenated Krebs solution. Synaptosomes were incubated with 50 nM [3H]-dopamine for 25 min, after which 1 ml aliquots of the suspension (∼0.35 mg protein) were injected into nylon syringe filters maintained at 36.5°C and superfused (0.4 ml/min) with preoxygenated Krebs. Under these superfusion conditions, spontaneous [3H]-dopamine efflux was essentially unaffected by reuptake. Sample collection (every 3 min) was initiated after a 20 min period of filter washout. Radioactivity in the samples and in the filter (at the end of experiment) was measured by liquid scintillation spectrophotometry.

Data, means ± SEM of 6–8 determinations per group, were calculated as absolute content (pmol/mg protein), fractional release (i.e. tritium efflux expressed as percentage of the tritium content in the filter at the onset of the corresponding collection period) or net fractional release, i.e. K+-evoked tritium overflow as percentage of the tritium content in the filter at the onset of the corresponding collection period. Statistical analysis was performed (Prism software) by one-way ANOVA followed by the Newman–Keuls test for multiple comparisons. When only two groups were compared, the Student t-test was used. P-values < 0.05 were considered to be statistically significant.

Microdialysis

Microdialysis experiments were carried out in awake, freely moving animals. Rats were anaesthetized, mounted in a stereotaxic frame (David Kopf Instruments) and implanted with a guide cannula (stainless steel, shaft outer diameter of 0.38 mm, length 4 mm; Metalant AB), in the hippocampus ipsilateral to the 6-OHDA-lesioned side (anterioposterior = −3.6; lateral = +1.84). Experimental procedures were performed as previously reported (Pascucci et al., 2007). Following the onset of perfusion, rats were left undisturbed for 2 h and then dialysates were collected at 20-min intervals for 3 h. Dialysate samples were transferred to high-performance liquid chromatography systems for biogenic amine detection. Both catecholamines were simultaneously measured at the following conditions: the conditioning cell was set at +400 mV, electrode 1 at +200 mV and electrode 2 at −250 mV; the mobile phase was described previously (Westerink et al., 1998). For 5-hydroxytryptamine detection, the conditioning cell was set at +350 mV, electrode 1 at −150 mV and electrode 2 at +200 mV; the mobile phase was described previously (Gartside et al., 2003). A Nova-Pack C18 column (3.9 × 150 mm; Waters) equipped with a Sentry Guard Nova-Pack C18 pre-column (3.9 × 20 mm) maintained at 32°C was used. The limit of sensitivity of the assay was 0.1 pg. The flow rate was 1.2 ml/min.

Tissue analysis

Tissue analysis was carried out as previously reported (Puglisi-Allegra et al., 2000). Briefly, following decapitation, the brain was dissected and put on an aluminium surface at 0°C. The punches of hippocampus were kept frozen and stored at −80°C. On the day of analysis, punches were weighed and homogenized in 0.05 M HClO4. Tissue levels of dopamine, norepinephrine, homovanillic acid and 3,4-dihydroxyphenylacetic acid (DOPAC) were assessed simultaneously by a high-performance liquid chromatography system.

Subcellular fractionation and western blot analysis

Purification of triton-insoluble postsynaptic fraction and western blot analysis were performed as previously reported (Gardoni et al., 2006). The following antibodies were used: polyclonal antibody anti-NR2B and monoclonal antibody anti-NR2A from Zymed Laboratories, monoclonal antibody anti-α-Tubulin from Sigma-Aldrich.

Electrophysiological recordings

Mice and rats were anaesthetized with halothane before decapitation. Under visual control, a stimulating electrode was inserted into the Schaffer collateral fibres, and a recording electrode was inserted into the CA1 region of the hippocampal slice (Sgobio et al., 2010). Field excitatory postsynaptic potentials (fEPSPs) were filtered at 3 KHz, digitized at 10 KHz and stored on a PC. For all of the experiments, data are presented as mean ± SEM (n is the number of slices). Off-line analysis was performed using Clampfit (Molecular Devices) and GraphPad Prism 3 (GraphPad Software) software. Two-way ANOVA was used for statistical analysis. The significance level was established at P < 0.05.

For patch-clamp recordings, neurons of the CA1 region were visualized using differential interference contrast (Nomarski) and infrared microscopy (Olympus). Whole-cell voltage-clamp (holding potential, −60 mV) recordings were performed with borosilicate glass pipettes. Postsynaptic currents (PSCs) of half-maximal amplitude were evoked every 10 s; LTP was induced by a high-frequency stimulation protocol consisting of three trains stimulating at same postsynaptic current strength. Details are given in the Supplementary material.

Results

6-OHDA injected into the rat medial forebrain bundle caused loss of dopamine neurons located in both the substantia nigra pars compacta and the ventral tegmental area (Fig. 1A). This procedure was accompanied by loss of the efferent nigral projections to the striatum and of the dopaminergic projections from the ventral tegmental area (P < 0.001). However, as shown in Fig. 1A and B, the dopaminergic loss was more evident in the substantia nigra pars compacta than the ventral tegmental area (P < 0.001) mimicking the pattern observed in Parkinson’s disease (Damier et al., 1999). In fact, while some dopamine neurons were spared in the ventral tegmental area, the dopamine denervation was virtually complete in the substantia nigra pars compacta.

Hippocampal-dependent learning is impaired in experimental parkinsonism: reversal by l-DOPA

In order to explore whether endogenous hippocampal dopamine is implicated in cognitive deficits observed in Parkinson’s disease, we measured the ability of both 6-OHDA-depleted and sham-operated rats (n = 10 for both groups) to recognize environmental spatial novelty by utilizing an open-field hole-board (Fig. 2A–C). This test has been demonstrated to involve the dorsal hippocampus and to be dopamine-dependent (Lemon and Manahan-Vaughan, 2006). In Session 1 (Fig. 2A and B), no significant differences in locomotor (horizontal and vertical) activities were observed between groups (P > 0.05). In the exposure of hole-board sessions (Fig. 2C), two-way ANOVA revealed a significant interaction between Group × Session main factors (P < 0.001). Post hoc analysis showed a significant reduction of hole explorations in sham-operated rats (P < 0.001) but not 6-OHDA-lesioned animals, indicating that parkinsonian rats have a recognition deficit of novel context feature (hole-board). Subchronic l-DOPA treatment administered (twice a day for four consecutive days) 4 h before test restored normal performance in lesioned rats (**P < 0.01; Fig. 2C).

To demonstrate that the behavioural effect induced by systemic l-DOPA was really dependent on the activation of hippocampal dopamine receptors, we injected the D1 receptor antagonist SCH23390 (1.5 µg/µl saline) in the hippocampus of dopamine-depleted (n = 21) and sham-operated rats (n = 7). SCH23390 or saline were injected (20 min before systemic l-DOPA administration) once a day for four consecutive days.

Intrahippocampal application of saline in sham-operated animals did not produce significant alterations in recognition ability of new context. Also, in hemiparkinsonian lesioned rats, cannula implantation and handling procedure for injection did not alter the deficit in recognition novelty and the capability of l-DOPA to restore habituation process. Interestingly, intrahippocampal delivery of SCH23390 fully prevented the l-DOPA-induced therapeutic effect in parkinsonian animals (Fig. 2C). This observation supports the critical involvement of hippocampal D1/D5 receptors in the observed behavioural effects induced by l-DOPA. Moreover, between-group post hoc comparisons revealed that sham-operated rats explored the hole-board significantly more than the other groups during first exposure (Fig. 2C). These results revealed an effect of 6-OHDA on basal arousal activity of the animals. l-DOPA treatment did not restore normal exploration activity suggesting that l-DOPA might fail to correct all the deficits caused by dopamine denervation and that non-dopaminergic systems might contribute to this deficit. Nonetheless, increasing brain dopamine with l-DOPA allows the animals to habituate to the hole-board in relation to their own level of arousal.

CA1 pyramidal neurons were patch-clamped in slices from hemiparkinsonian (n = 9) and sham-operated (n = 6) rats. The current–voltage relationship revealed no differences in basal membrane properties between neurons recorded in slices from sham-operated and 6-OHDA-lesioned rats (Fig. 3A; P > 0.05). Postsynaptic currents (PSCs) and extracellular field potential (field EPSP) recordings were subsequently obtained from hippocampal slices taken from hemiparkinsonian and sham-operated rats (n = 20 and n = 8 for field EPSP recordings, respectively). At the beginning of each experiment, an input–output curve was obtained by stimulating the collateral Schaffer fibres and recording from the CA1 region of the slice. The comparison of the curves obtained from 6-OHDA and sham-operated slices revealed no significant difference between groups for both the postsynaptic current (Fig. 3B and C; n = 4 for each group, P > 0.05) and for the field EPSP (n = 8 slices for each group).

We investigated the possibility to restore hippocampal LTP with l-DOPA. Bath application of 30 µM l-DOPA was able to rescue LTP, as shown by recovery of field EPSP potentiation (Fig. 4A; n = 9 slices for each group; P < 0.001). Interestingly, l-DOPA did not affect LTP amplitude in the hippocampus of sham-operated rats (Supplementary Fig. 1A) or in the contralateral hippocampus of 6-OHDA-lesioned rats (Supplementary Fig. 1B).

l-DOPA restores long-term potentiation in the hippocampus of 6-OHDA-lesioned rats by its conversion into DA activating D1 receptors. (A) Traces and time-course plots of field EPSP (fEPSP) recorded in the standard solution, in the presence of 30 µM l-DOPA and in the presence of 30 µM l-DOPA plus 100 µM carbidopa. Note the enhanced LTP in the presence of l-DOPA but not in the presence of l-DOPA plus carbidopa; [161.3 ± 5.0%, and 123.2 ± 7.0% in the presence and the absence of l-DOPA, respectively, n = 9 field EPSPs for each group, F(80,920) = 1.82; ***P < 0.001]. (B) Traces and time-courses of field EPSPs from slices of 6-OHDA lesioned rats and of 6-OHDA rats subchronically treated with l-DOPA [intraperitoneal; field EPSPs potentiation after systemic l-DOPA treatment, 166.8 ± 9.5% versus field EPSPs potentiation in untreated hemiparkinsonian animals 123.2 ± 7.0%, respectively; n = 6 for l-DOPA-treated rats and n = 9 for 6-OHDA rats, F(40,520) = 5.97; ***P < 0.001]. (C) The traces and time-course plots show field EPSPs recorded in the standard solution and in the presence of 30 µM l-DOPA plus the D1 DA receptor antagonist SCH23390 (SCH, 10 µM). (D) Traces and time-course of field EPSPs recorded in the standard solution, in the presence of the D1 DA receptor agonist SKF38393 (SKF, 10 µM) [155.5 ± 1.9%, n = 6 field EPSPs, F(80,720) = 5.93; ***P < 0.001] and in the presence of the D2 DA receptor agonist quinpirole (10 µM; P > 0.05). HFS = high-frequency stimulation.

To test the hypothesis that l-DOPA effects were dependent on its conversion to dopamine, we performed experiments in the presence of the DOPA-decarboxylase inhibitor carbidopa. l-DOPA (30 µM) plus 100 µM carbidopa were bath applied 20 min prior to the induction of LTP and throughout the experiment. In these conditions, LTP was not different from that observed in untreated 6-OHDA slices (Fig. 4A; n = 8 slices, P > 0.05).

To confirm the dopamine-dependence of hippocampal LTP, we analysed LTP in slices obtained from 6-OHDA-lesioned animals subchronically treated with l-DOPA. Similar to the acute effect of l-DOPA, LTP was restored in l-DOPA treated animals (Fig. 4B; n = 6 for l-DOPA-treated rats and n = 9 for 6-OHDA rats; P < 0.001).

To investigate the dopamine receptor subtype involved in l-DOPA action, we applied 30 µM l-DOPA in the presence of the D1/D5 receptor blocker SCH23390 (10 µM). In this condition, l-DOPA failed to restore LTP, suggesting that the action of l-DOPA was due to its conversion to dopamine acting on D1/D5 receptors (Fig. 4C; l-DOPA plus SCH23390, n = 5 versus control in the standard solution, n = 9 slices, P > 0.05). In slices obtained from dopamine-denervated animals, SCH23390 failed to decrease the LTP amplitude further (Supplementary Fig. 1C).

Moreover, we used high-performance liquid chromatography to measure tissue level of endogenous dopamine content in the hippocampus of α-syn120 (n = 3) and wild-type (n = 3) mice. No difference in dopamine content was found between α-syn120 and control mice (Fig. 5D; P > 0.05) whereas a decrease in homovanillic acid (a dopamine metabolite) was detected in α-syn120 mice (Fig. 5E; P < 0.01). Conversely, the levels of DOPAC (the metabolite generated by monoamine oxidase) did not change (data not shown). This evidence should be interpreted as a decrease in dopamine turnover associated with a possible catechol-O-methyltransferase (COMT) defect in the hippocampus of this genetic Parkinson’s disease model. In addition, α-syn120 mice presented a slight reduction in norepinephrine (Fig. 5F; P < 0.05), but no difference in 5-hydroxytryptamine levels (Fig. 5G; P > 0.05) compared with control mice, which may point to the role of others catecholaminergic neurotransmitters in cognitive disturbances in these mice.

Altered distribution of NMDA receptor subunits in 6-OHDA-lesioned and in α-syn120 transgenic animals. Hippocampal Triton-insoluble fractions (TIF) from parkinsonian and control animals were analysed by western blot analysis with antibodies for NMDA receptor NR2A and NR2B subunits. The same amount of protein was loaded per lane. Histograms show the quantification of western blotting performed in hippocampal TIF from 6-OHDA and sham-operated rats (A) and from α-syn120 transgenic and wild-type mice (WT) (B); (*P < 0.05, **P < 0.005).

We also evaluated whether the LTP impairment in α-syn120 transgenic mice (n = 5) was correlated with changes in NMDA receptor composition in the postsynaptic compartment. Interestingly, a decrease in NR2A (−30.7 ± 6.7%, P < 0.05 compared with wild-type, n = 5) and a concomitant decrease in the NR2A/NR2B ratio (−35.2 ± 4.9%, P < 0.05 compared with wild-type) were detected in α-syn120 transgenic mice compared with wild-type mice (Fig. 7B). These findings demonstrate that, although toxic and genetic models of Parkinson’s disease show distinct neurochemical and molecular patterns, they can share similar alterations in the NR2A/NR2B ratio, possibly leading to a reduced hippocampal LTP.

In order to investigate the role of NMDA receptor subunits on CA1 hippocampal LTP recorded in sham-operated and 6-OHDA denervated animals (n = 5 for each group), we analysed synaptic plasticity in the presence of ifenprodil, an antagonist of NR2B receptor subunit. Ifenprodil (1 µM) significantly reduced the LTP observed in sham-operated animals (Fig. 8A; n = 5, P < 0.001), while it did not affect the LTP amplitude in dopamine-denervated rats (Fig. 8B; n = 4, P > 0.05). Previous studies have shown the capability of cell-permeable TAT peptides fused to the C-terminal domain of NMDA receptor subunits to reach and to disrupt NMDA/PSD-MAGUKs (PSD-95-like membrane associated guanylate kinases) association both in in vitro and in vivo studies (Aarts et al., 2002; Gardoni et al., 2006). Thus, we also analysed LTP in the presence of the cell-permeable peptides TAT2A and TAT2B, respectively, and selectively targeted NR2A and NR2B subunits of NMDA receptor in both sham-operated and 6-OHDA denervated rats. In these experiments, slices were incubated with either TAT2B or TAT2A before (at least 2 h) and during the electrophysiological recordings. The application of 300 nM TAT2B peptide significantly decreased LTP in sham-operated animals (Fig. 8C; n = 3, P < 0.001), as previously reported for CA1 hippocampal LTP in control animals (Gardoni et al., 2009). However, this peptide, in sharp difference with ifenprodil, fully restored LTP to control levels in 6-OHDA denervated rats (Fig. 8D; n = 5, P < 0.001) suggesting that the correct assembly of the NMDA receptor subunits, rather than their pharmacological blockade, is necessary to restore physiological plasticity. In line with this observation, we also found that the TAT2A peptide significantly decreased CA1 LTP in sham-operated animals (Fig. 8E; n = 8, P < 0.001) but not in dopamine-denervated animals (Fig. 8F; n = 4, P > 0.05), confirming the molecular data and further supporting the hypothesis that a correct balance between NR2A and NR2B is critically important for LTP induction.

Discussion

In the present study, we have shown that CA1 hippocampal LTP is reduced in both a neurotoxic and a genetic model of Parkinson’s disease. This plastic alteration is associated with neurochemical changes of dopamine transmission, deficits of hippocampal-related memory tasks and abnormalities in the expression of hippocampal NMDA receptor subunits. We achieved these results by analysing, for the first time, the role of hippocampal synaptic plasticity in both a toxic and a genetic model of Parkinson’s disease using combined electrophysiological, behavioural, molecular and neurochemical approaches. The 6-OHDA-induced lesion is still the most widely used model for replicating a Parkinson’s disease-like loss of nigral dopaminergic neurons and it is currently adopted for the analysis of striatal synaptic plasticity and its link with motor symptoms. Surprisingly, this toxic model has been less utilized to investigate altered synaptic plasticity in other brain areas such as hippocampal LTP and the correlated memory deficits. On the other hand, the genetic model used in the present study expresses truncated human α-synuclein (1–120), leading to the formation of pathological inclusions in the substantia nigra pars compacta and olfactory bulb and to a reduction in striatal dopamine levels. At the behavioural level, transgenic mice show a progressive reduction in spontaneous locomotion mimicking the pathological and clinical features of Parkinson’s disease (Tofaris et al., 2006). However, hippocampal dopamine transmission and plasticity and their possible involvement in memory deficits have never been deeply investigated in genetic models.

Two major findings suggest that the reduction of endogenous hippocampal dopamine plays a major role in the decrease of LTP in experimental parkinsonism. First, in the 6-OHDA denervated hippocampus the release of dopamine during membrane depolarization is significantly reduced in comparison with control animals. Secondly, in both neurotoxic and genetic models of Parkinson’s disease, the administration of l-DOPA, a dopamine precursor, restores a physiological LTP. The effect of l-DOPA is blocked by hippocampal administration of a DOPA-decarboxylase inhibitor such as carbidopa, further supporting the idea that l-DOPA does not exert its therapeutic action on hippocampal synaptic plasticity per se but only after its conversion to dopamine.

In our 6-OHDA-induced lesioned model of Parkinson’s disease, the dopaminergic loss was complete in substantia nigra pars compacta while some dopamine neurons were spared in the ventral tegmental area. This condition partially mimics Parkinson’s disease where there is a more severe loss of dopamine cells in substantia nigra pars compacta than in ventral tegmental area. However, a significant cell loss in the ventral tegmental area has been observed in Parkinson's disease brains (Uhl et al., 1985; German et al., 1989).

Interestingly, an impaired hippocampal LTP and an altered hippocampal-dependent memory were also observed in a transgenic mouse model for α-synuclein aggregation obtained by the expression of human α-syn120 under the control of the tyrosine hydroxylase promoter (Tofaris et al., 2006). These findings do not match with a previous observation in which no difference in hippocampal LTP was reported in a transgenic mouse line carrying the α-synuclein A30P mutation (Steidl et al., 2003). These data, however, were obtained in a mouse line showing normal dopamine levels, dopamine receptor number and dopamine function, features that are importantly different from those of the α-syn120 model, where dopamine signalling was affected (Tofaris et al., 2006; Garcia-Reitbock et al., 2010).

The absence of motor abnormalities observed in mutant mice in our study is in line with a previous study showing that, in these mice, spontaneous locomotor activity was altered at 18 but not at 6 months of age (Tofaris et al., 2006). Thus, at least in this genetic model, deficits in hippocampal function seem to precede motor symptoms.

Unlike patients with Parkinson’s disease and 6-OHDA rats, α-syn120 mice do not show significant neuronal cell death (Tofaris et al., 2006). Thus, impairment of dopamine transmission in hippocampus (present study) and striatum (Tofaris et al., 2006) cannot be attributed to dopamine cell loss but more likely to functional changes of the release machinery caused by the early accumulation of α-syn120 in presynaptic terminals. Accordingly, alterations in dopamine release associated with redistribution of SNARE proteins have recently been described in the striatum of these mice (Garcia-Reitbock et al., 2010).

The hippocampus represents a key structure for the formation of spatial and episodic memories (Ryan et al., 2010) as well as for the detection of novelty (Jenkins et al., 2004). This structure is critically involved in the storage of information such as memory for places (Muzzio et al., 2009). In line with this view, in the present study we found that habituation to a novel environment is altered in hemiparkinsonian animals bearing impaired hippocampal LTP, and that these behavioural and biochemical deficits, as well as abnormalities in synaptic plasticity, can be reversed by systemic l-DOPA. Thus, our data provide a novel link among spatial memory, hippocampal LTP and neurochemical changes underlying Parkinson’s disease.

The ventral tegmental area/hippocampal dopaminergic loop regulates the flux of information into long-term memory and this system is believed to play an important role in information acquisition and synaptic plasticity (Lisman and Grace, 2005). It has been proposed that the CA1 neurons could detect mismatches between predictions from the dentate gyrus–CA3 network and sensory input from the cortex (Lisman and Otmakhova, 2001). The behavioural function exerted by the CA1 hippocampal area could be of critical importance since novelty detection involves the comparison of an existing memory with new sensory information.

Our data allow the establishment of an intriguing association between the roles of D1/D5 receptors in hippocampal synaptic plasticity and cognitive dysfunction in Parkinson’s disease. In fact, although both l-DOPA (acting on all the different types of dopamine receptors after its conversion to dopamine) and D2 receptor agonists are widely used in patients with Parkinson’s disease to improve motor function, they cause different modulation on neuropsychological functions (Robbins and Arnsten, 2009).

Several studies have suggested that NR2A- and NR2B-containing receptors have different roles in the regulation of the induction of LTP in the hippocampus. Enhanced expression of NR2B seems to facilitate the induction of LTP (Tang et al., 1999; Foster et al., 2010). It has been postulated that the NR2B subunit plays a critical role for LTP, presumably by recruiting relevant molecules important for LTP via its cytoplasmic tail (Foster et al., 2010). In contrast, NR2A does not seem to be critical for the LTP and its cytoplasmic tail seems to carry inhibitory factors for LTP (Foster et al., 2010). Accordingly, hippocampal LTP is severely impaired by experimental approaches selectively targeting NR2B NMDA subunit, such as selective NR2B antagonists (Bartlett et al., 2007), antisense knockdown of NR2B (Clayton et al., 2002) and cell-permeable peptides reducing the NR2B localization to synaptic sites (Gardoni et al., 2009). Conversely, Liu et al. (2004) showed that NR2A but not NR2B subunits are required for hippocampal LTP. Differences in the experimental approaches used might possibly account for the obtained conflicting results. However, an emerging concept derived from all these studies is the requirement of a correct balance in the NR2A/NR2B subunit composition at hippocampal synapses as a critical condition for LTP induction (Liu et al., 2004; Bartlett et al., 2007; Bellone and Nicoll, 2007; Morishita et al., 2007; Gardoni et al., 2009). In agreement with these studies, here we show that, although a toxic and a genetic model of Parkinson’s disease both show distinct neurochemical and molecular changes, they can induce a significant decrease of NR2A/NR2B subunit ratio in hippocampal synaptic NMDA receptors possibly leading to LTP impairment and memory dysfunctions. These molecular findings are also supported by our electrophysiological experiments showing that, while the TAT2B peptide reduces LTP in sham-operated animals showing a normal NR2A/NR2B ratio, in 6-OHDA denervated rats it restores this form of synaptic plasticity to a control level. These molecular and electrophysiological findings suggest that changes in the NR2A/NR2B ratio at CA1 hippocampal synapses might be a key factor to explain both plastic and cognitive dysfunction in Parkinson’s disease.

Supplementary material

Acknowledgements

We wish to thank Dr Robert Nisticò for critical reading of the manuscript and suggestions and Mr. Cristiano Spaccatini for his excellent technical support. We are grateful to Dr Oleg Anichtchick for help and advice with the α-synuclein (1–120) transgenic mice.